Numerous studies examining atherosclerotic lesions from human and animal models have established the central role of the macrophage in atherosclerosis. Despite these observations, it is still unclear how the multiple pro- and anti-inflammatory capabilities of the macrophage are balanced within lesions. One potentially important mechanism for them to regulate their function is by the rapid modulation of the repertoire of proteins expressed on their cell surface through proteolytic "shedding". In addition to dynamically altering the cell surface constituents, shedding also leads to the release of soluble ectodomains with distinct biological properties. This proposal will focus on the ADAM family of proteases that have gained recognition as primary effectors of ectodomain shedding. Early lesions of atherosclerosis are characterized by lipid-filled macrophages. Scavenger receptors are responsible for this massive accumulation of cholesterol, and their significance for atherogenesis is highlighted by multiple gene knockout studies. Fas ligand (FasL) is a key cell surface regulator of macrophage apoptosis and activation. Both scavenger receptors and FasL can be proteolytically cleaved from the cell surface resulting in down-regulated cellular expression. The release of soluble scavenger receptor can inhibit foam cell formation in vitro and in vivo, while FasL is proteolytically released in both active and inactive forms. Thus, proteolytic shedding of scavenger receptors and FasL could modulate lesion initiation and progression. However, the enzymes responsible for their shedding have not been fully characterized. In Aim 1, we will determine the proteases involved in the shedding of scavenger receptors and FasL, and the functional significance of shedding on atherogenesis will be examined in Aim 4 by expressing uncleavable mutants of these substrates. In addition to possible effects on lesion initiation, ectodomain shedding may also contribute to lesion progression and plaque rupture. Macrophage activation induces the shedding a multitude of inflammatory mediators, and ADAM17 has been shown to be responsible for the shedding of several of these. In Aim 4, we will test the role of ADAM17 in lesion initiation, progression and plaque rupture by genetically modulating its expression in macrophages. In addition, proteomic approaches will be utilized to identify novel substrates for ADAM17 potentially involved in atherosclerosis (Aim 2), and to determine the role played by oxidants in regulating the activity of this enzyme (Aim 3) in collaboration with Project 4.